Blood plasma proteins serve a wide variety of functions in the human body such as the maintenance of blood volume, osmotic pressure, viscosity, and other important physical parameters of blood. A number of commercial processes have been implemented to separate and purify these proteins from blood plasma for therapeutic use. Some common methods for protein purification include precipitation with ammonium sulfate and similar salts, organic solvent precipitation with cold ethanol or acetone and other such alcohols and ketones, selective adsorption on gels, isoelectric precipitation, and chromatography by use of adsorbents. Still other processes for selectively fractionating and purifying blood proteins involve the use of amino acids, water-soluble organic polymers, and water-insoluble polyelectrolyte polymers containing basic amino groups.
Many protein extraction and purification techniques are based on altering the solubility of a desired protein in a biologic fluid such as blood plasma or a plasma solution by adjusting any of number of properties of the protein solution. Through the addition of salts or dilution of a solution, separation may be carried out in the range of low ionic strengths at which the interactions of proteins with electrolytes differ from each other, both in the isoelectric condition and when dissociated as acids or bases. The solubility of a protein may also be reduced by the addition of alcohols, acetone or other water miscible organic solvents to protein solutions. The balance between the precipitating action of alcohols and the salt interactions permits attainment of a variety of conditions under which the protein to be separated may be brought to a desired solubility. This balance may be altered based on the pH and temperature for each protein component; however, to avoid denaturing of the desired protein, sufficiently low temperatures should be maintained. Moreover, the pH may be controlled by adding a buffer such as an acetate or other buffers of known ionic strength, and adjusted so as to take advantage of the differences in the isoelectric points and the directions of the interactions with salts of the protein components to be separated. Finally, the concentration of protein may be maintained as low as possible to obtain a desired amount of protein precipitate to minimize protein-protein interactions. As the number of components in the solution or suspension increases or if multiple components have similar physical chemical properties, more of these variables must be accurately controlled to lower the solubility of a single protein type.
Because these extracted and/or purified plasma proteins may be used therapeutically in humans, such extraction and purification processes require rigorous quality control, such as monitoring and analytical testing of the biologic fluid to ensure that the end-product is both consistent and safe, and that the chemical properties of the mixture are kept consistent with the intended process design. For example, conventional monitoring techniques such as the use of enzyme-linked immunosorbent assays (“ELISAs”) or Surface Plasmon resonance may be used to determine the amount of antibody activity in a prepared sample. Other common monitoring methods such as reversed-phase, affinity or cation-exchange chromatography require the preparation of samples and controls and incubating for a certain period of time to monitor protein degradation. Additionally, SDS-Page electrophoresis techniques can be used to determine antibody impurities.
Unfortunately, many of these known monitoring and control techniques are costly and may require specialized antigens, reagents and equipment to perform the requisite analyses. In fact, many of these known techniques are unsuitable for monitoring and controlling separation processes in real-time, as they require sample preparation, specific incubation times and/or other time-consuming steps.
A need therefore exists for Process Analytical Technology (PAT) methods and systems for in-process monitoring of suspensions and solutions in an improved manner that may enhance protein process understanding, improve process control, and/or achieve consistent product quality. As used herein, PAT includes, for example, a system for designing, analyzing, and/or controlling manufacturing through timely measurements (i.e., during processing) of critical quality and performance attributes of raw and in-process materials and processes with the goal of ensuring final product quality. It would be beneficial if these desired attributes were obtainable using a method that is fast, reliable, low maintenance and involves an ease in the use of equipment, such as the use of equipment that does not need to be dismantled or the use of sensors that are easily cleaned.